【作者】 徐杨；

【导师】 黄晓峰； 孙国文；

【作者基本信息】 南京大学 ， 口腔临床医学， 2016， 硕士

【摘要】 [目的]HPV相关口腔鳞癌(OSCC)代表一种独特的生物学和临床疾病,OSCC Ⅰ临床样本HPV感染的高效精准检测有助于判断肿瘤分期和患者预后,从而指导制定最有利于患者的治疗方案。p16可作为宫颈癌和口咽鳞癌HPV感染的替代标记物,但其在OSCC中意义未知。研究p16在OSCC中的表达及临床病理学意义,分析OSCC中p16表达和HPV感染的相关性及对预后影响,探讨OSCC HPV检测的综合性策略。[方法]收集南京大学医学院附属南京市口腔医院2013年2月-2015年1月确诊并收入于生物样本库的217例OSCC,l临床病理资料完整,随访结果齐全。采用免疫组织化学染色(IHC)检测p16、p53、Ki67在217例OSCC中的表达,分析其临床病理学意义。同时选取p16阳性细胞数比例>25%的30例OSCC样本,通过逆转录定量聚合酶链式反应(RT-qPCR)和PCR-反向点杂交法检测HPV感染情况。[结果]1、IHC检测p16在217例OSCC中的表达,30例p16阳性细胞数比例>25%,16例p16 IHC阳性细胞数比例>70%。RT-qPCR检测30例p16阳性细胞数比例>25%的OSCC中HPV感染,3例为HPV16阳性,2例为HPV18阳性。PCR-反向点杂交法检测30例p16阳性细胞数比例>25%的OSCC中HPV基因分型,1例为HPV16阳性,1例为HPV82阳性。HPV感染与p16阳性表达呈显著正相关(P<0.05),HPV感染的OSCC均为p16阳性细胞数比例>70%,但并非所有p16阳性细胞数比例>70%的OSCC都有HPV感染。2、217例OSCC中,p16阳性表达率为7.4%,在OSCC病变部位上有显著性差异(P<0.05)；p53阳性表达率为59.4%,在OSCC Ⅰ级组显著高于OSCC Ⅱ级组(P<0.05)；Ki67阳性表达率为32.3%,在OSCCN1/N2组显著高于OSCCNO组(P<0.05),在OSCC Ⅱ级组显著高于OSCC Ⅰ级组(P<0.05)；HPV感染在OSCC性别组、年龄组、病变部位组、T分期组、N分期组及组织学分级组均无显著性差异(P>0.05)。p16表达与p53表达呈负相关,有统计学意义(P<0.05)；p16表达与Ki67表达有负相关趋势,但无统计学意义(P>0.05)Ki67阳性表达组OSCC预后差于Ki67阴性表达组OSCC,有统计学差异(P<0.05)；p16表达、p53表达和HPV感染与OSCC预后无显著相关性(P>0.05)。p16 (+)/HPV (+)组OSCC患者预后最好,有统计学意义(P<0.05)；P16/p53联合表达、p16/Ki67联合表达则与OSCC预后无显著相关性(P>0.05)。[结论]1、p16不能独立作为检濒OSCC HPV感染的替代标记物,但可用于OSCC HPV感染的初筛检测,当p16 IHC阳性细胞数比例>70%时应补充更严格的HPVPCR或HPV ISH。2、本组OSCC中,p16表达与病变部位有关；p53表达与组织学分级关系密切；Ki67表达与淋巴结转移和组织学分级关系密切；HPV感染则与各临床病理学参数无关。p16表达与p53表达呈负相关。Ki67阳性表达有较差的预后；p16表达、p53表达和HPV感染则对预后无影响。p16 (+)/HPV(+)患者预后最好；p16/p53联合表达、p16/Ki67联合表达则与预后无关；p16/HPV联合表达可作为判断OSCC预后的一个有用指标。更多还原

【Abstract】 [Objective]HPV related oral squamous cell carcinoma (OSCC) represents a unique biological and clinical disease. Efficient and accurate detection of HPV in OSCC clinical samples is helpful to judge tumor stage and prognosis, so as to guide the best treatment for patients. p16 can be used as a surrogate marker of HPV infection in cervical cancer and oropharyngeal squamous cell carcinoma. But its significance in OSCC is unknown. We aim to study the expression of p16 and its clinicopathological significance in OSCC, and analyze the correlation between p16 expression and HPV infection and their influence on the prognosis of OSCC patients. Thus we will explore the comprehensive strategy of HPV detection in OSCC.[Method]217 cases of OSCC which had complete clinicopathological data and follow-up results were obtained from the biological sample library at Nanjing Stomatology Hospital, Medical School of Nanjing University between February 2013 to January 2015. We detected the expression of p16, p53, Ki67 in 217 OSCC samples by IHC. Then we selected the cell staining of p16>25% smaples to detect HPV by reverse transcription quantitative polymerase chain reaction(RT-qPCR) and PCR-reverse dot blot method.[Results]1. In 217 OSCC smaples, there were 30 with p16 staining> 25% and 70 with p16 staining> 75%. Among the 30 p16>25% samples, we detected 5 HPV positive smaples. They were further catogerized into HPV 16 (3 cases) and HPV18 (2 cases) by RT-qPCR. Among the same 30 cases, we detected 2 HPV positive smaples by PCR-reverse dot blot method, among them 1 was HPV16 and 1 was HPV82. The infection of HPV was positively correlated with the expression of p16 (P<0.05). All the HPV positive cases had more than 70% p16 positive cells, but not all p16 positive cell number>70% cases were HPV-positive.2. Among 217 OSCC samples,7.4% were p16 positive. The expression of p16 was significantly correlated with lesion sites (P<0.05). There were 59.4% p53 positive cases. The expression of p53 was significantly higher in OSCC I group than in OSCC Ⅱ group (P<0.05). There were 32.3% Ki67 positive cases. It was increased in N1/N2 group compared with NO group (P<0.05). The expression of Ki67 was significantly higher in OSCC Ⅱ group than in OSCC I group (P<0.05). Whether HPV infected or not was not correlated with sex, age, sites, TNM or histological grade (P> 0.05). The expression of p53 was negatively correlated with the expression of p16 (P <0.05). There was a negative correlation trend between the expression of p16 and Ki67, but without statistical significance (P>0.05). The prognosis of OSCC patients was significantly worse in Ki67 positive group than that in Ki67 negative group (P< 0.05). HPV infection and the expression of p16, p53 were not significantly related to the prognosis of OSCC patients (P> 0.05). Meanwhile, p16(+)/HPV(+) OSCC patients had the best prognosis (P<0.05). The combination of p16 and p53 or p16 and Ki67 had no correlation with the prognosis of OSCC patients (P>0.05).[Conclusion]1. p16 could not serve as a surrogate marker for HPV status in OSCC independently. However, p 16 IHC could be used to detect the initial screening of HPV infection in OSCC. If there are more than 70% p16-positive cells in IHC, we should apply HPV PCR or HPV ISH to determine HPV infection.2. In our OSCC samples, the expression of p16 was significantly related to the lesion site. The expression of p53 was closely related to the histological grade. The expression of Ki67 was closely related to lymph node metastasis and histological grade. The status of HPV was not correlated with clinicopathological parameters. The expression of p16 was negatively correlated with the expression of p53. The Ki67 positive patients have poorer prognosis. The expression of p16 and p53 and HPV infection had no effect on prognosis. p16(+)/HPV(+) OSCC patients had the best prognosis. The combination of p16 and p53 or p16 and Ki67 had no correlation with the prognosis of OSCC. The combination of p16 and HPV may be a useful index to judge the prognosis of OSCC.更多还原